Project Summary The clinical use of anti-androgens such as abiraterone and enzalutamide has greatly improved prostate cancer treatment, but patients treated with these drugs still relapse with more aggressive forms of the disease, collectively termed as castration-resistant prostate cancer (CRPC). These forms of CRPC are characterized by increased lineage plasticity, often associated with loss of androgen receptor (AR) expression and neuroendocrine differentiation. Our laboratory focuses on analyses of cell type differentiation in the normal and transformed prostate epithelium, and has recently used genetically-engineered mouse models to show that neuroendocrine cells in CRPC arise by transdifferentiation from luminal adenocarcinoma cells. In preliminary studies for this proposal, we have generated organoid models of CRPC from these mouse prostate tumors, and have demonstrated by single-cell RNA sequencing that these organoids recapitulate much of the spectrum of human CRPC, including distinct heterogeneous populations composed of AR-pathway positive prostate cancer, neuroendocrine prostate cancer, and double-negative prostate cancer. Further analysis of these organoid lines has revealed a complex genomic landscape of chromatin accessibility, and has identified active histone marks that are associated with neuroendocrine differentiation. These findings indicate that epigenetic reprogramming may play a key role in the lineage plasticity of castration-resistant prostate cancer. Based on these preliminary data, we hypothesize that molecular analysis of epigenetic reprogramming in castration-resistant prostate cancer will identify candidate drivers and mechanisms of lineage plasticity. To investigate this hypothesis, we will pursue three innovative aims that integrate in vivo, ex vivo, molecular, and computational systems approaches to analyze genetically-engineered mouse models, organoids, grafts, and human prostate tumor samples. Our specific aims are as follows: (1) Analysis of lineage plasticity in CRPC organoid and mouse models to examine potential pathways of interconversion between distinct forms of CRPC; (2) Investigation of epigenetic pathways in CRPC organoid models by examining chromatin accessibility, histone marks, and DNA methylation patterns to identify epigenetic marks and regulators that drive lineage plasticity; and (3) Functional analysis of candidate regulators of lineage plasticity in CRPC using computational systems approaches to identify candidate regulators of plasticity followed by experimental validation using organoid and graft assays together with analyses of human tumor samples. Overall, these studies will provide essential insights into the molecular basis of lineage plasticity and treatment resistance in prostate cancer, and will have significant implications for the development of novel therapies.